Application of Mammalian Cells

Question 1

What is an antibody?

Answer 1

A naturally occurring protein used by the immune system to identify and neutralise foreign objects (eg bacteria, viruses)

Each antibody recognises a specific antigen unique to its target

Question 2

What is an antigen?

Answer 2

Any substance that causes the immune system to produce antibodies against it

Question 3

What is an epitope?

Answer 3

The specific region in the antigen that is recognised by the immune cells

Question 4

Name the two antibody types and explain them

Answer 4

Monoclonal (MAb)
- identical antibodies produced by one type of immune cell
- expensive can be produced in large quantities - infinite supply
- bind to same epitopes on taerget antigen

Polyclonal
- derived from different cell lines, mixture of antibodies
- cheaper but limited supply
- bind to same antigen but different epitopes

Question 5

What are the 2 major functions of an antibodies?

Answer 5

1) to recognise and bind to antigens

2) to induce immune response after binding

Question 6

What are the regions of an antibody and what do they do ?

Answer 6

The variable region mediates binding

The affinity for a given antigen is determined by the variable region

The constant region mediates the immune response after binding

Question 7

What are the historical milestones associated with antibodies?

Answer 7

1975 - cesar milstein and George’s kohler : the hybridoma technique

1984 - milatein and kohler received Nobel prize

1986 - first mouse monoclonal antibody approved for human use ( murimonav - cd3- immunosuppressant for organ transplant)

2003 - first human monoclonal antibody (adalimumab = humira- for rheumatoid arthritis

Question 8

Explain the invivo and in vitro method of the hybridoma technique

Answer 8

In vivo : injected in mice ( in peritoneal cavity, gut) produce tumours containing antibodies rich fluid (ascites fluid)

In vitro : grown indefinitely in cell culture media

Question 9

Give the advantages and disadvantages of the in vivo method

Answer 9

ADV
- cheaper
- higher MAb production yield (5-20mg/ml)

DISADV
- unethical
- requires specialised personnel (specialised license for handling animals

Question 10

Give the advantages and disadvantages of the in vitro method

Answer 10

ADV
- ethical
- doesn’t require specialised personnel

DISADV
- lower MAb productivity (5-10ug/ml)
- some hybridoma lines don’t grow in culture

Question 11

Why are hybridoma cells micro encapsulated in alginate capsules?

Answer 11

This is because it significantly increases the MAb yields (10-100ug/ml) as a result of higher cell density

Damon biotech company and cell tech use encapsulated hybridoma cells for large scale production of MAbs. They employ 100 litre fermenters to yield about 100g of MAbs in about 2 week period

Question 12

What are the disadvantages of the hybridoma technique?

Answer 12

• laborious
• expensive
• time consuming
• often caused immune reactions in patients

Question 13

Suggest an alternative technology for MAb production

Answer 13

Recombinant DNA Technology

Question 14

What is recombinant DNA technology ?

Answer 14

The insertion of DNA molecules/particles from a different species into a host organism (expression system) to produce useful products

Question 15

Explain the basic principles of recombinant dna technology

Answer 15

When a fragment of dna from a donor cell or organism is isolated and then inserted into the dna of another cell or organism

This allows scientists Toni traduce a new characteristic into and organism by inserting a new gene into it

Recombinant dna tech involves 4 steps
1) a dna fragment containing the gene of interest is obtained from donor cell
2) a suitable plasmid is obtained from a bacterium
(Plasmids are commonly used as vectors to transfer the gene of interest into the host cell for expression
3) the dna fragment containing the gene of interest is cut using an enzym called restriction enzyme (enzyme recognises a specific base sequence and cures the dna at a specific point)
Same restriction enzyme is used to cut the plasmid
4) the dna fragment containing th green of interest is inserted into the open plasmid with the help of another enzyme called dna ligase (acts like a glue)
Calatlyses the jointing of dna fragment and plasmid

A recombinant plasmid is formed
The plasmid can be introduced to a host cell for different purposes for example producing protein or GM fruit and veg

Question 16

Give 3 examples of expression vectors

Answer 16

• plasmid DNA
• virus vector
• bacterial vector

Question 17

Give 2 examples of expression systems

Answer 17

Prokaryotic - bacteria ( ecoli)

Eukaryotic
- yeast ( sacharomyces verrvisiae)
- viral (baculovirus)
- mammalian ( CHO)

Question 18

What expression system would be used for the following applications : large protein, small proteins, glycosylation, high yield low cost and post translational modifications?

Answer 18

Large proteins (>100 KD ) - eukaryote

Small protein (<30kD) - prokaryote

Glycosylation - baculovirus or mammalian cell culture

High yield low cost - E. coli

Post transitional modifications - yeast or baculovirus or mammalian cells

Question 19

What are the advantages and disadvantages of mammalian cells (eg CHO, HEK293) as expression systems?

Answer 19

Adv
- increased level of expression
- production of native structure proteins (correctly folded with appropriate bonding)
- easy to scale up by fermentation

Disadv
- expensive
- slower growing

Question 20

What are the advantages and disadvantages of bacterial cells (eg E.coli) as expression systems?

Answer 20

Adv
- Simple, well understood genetics
- easy to manipulate genetically
- very cheap
- fast expression with short doubling time
- easy to scale up by fermentation

Disadv
- protein folding issue resulting in insoluble inclusion bodies
- low yield for large proteins
- glycoprotein modification issues

Question 21

What is transformation in terms of DNA?

Answer 21

The alteration of genetic composition of a cell via the uptake of foreign DNA
- naturally I’m bacteria bu conjunction ( intra or inter species)
- naturally in bacteria by transduction ( viral- medicated DNA incorporation)
- artificially by chemical method and by electroporation

Question 22

What is transfection?

Answer 22

The transformation and infection of cells to introduce foreign DNA

Question 23

List 6 properties of transient transfection

Answer 23

• No genomic integration
• short term expression (24-48h)
• DNA is degraded by nucleuses or dilutes during cell division
• useful for rapid analysis of phenotype or for recombinant protein production
• works best with supercooled circular plasmids
• chemical method or electroporation

Question 24

List 6 properties of stable transfection

Answer 24

DNA is integrated into the cells genome and is replicated with the host DNA

Permanent

Sustained expression for long periods of time

Works best with viral DNA

Viral or micronisation

Question 25

Explain the process of transfection

Answer 25

Rely on masking the negatuve charges of the phosphate backbone of DNA to facilitate precipitation/ cell uptake Particulate DNA complexes are taken up into cells by endocytosis Naked DNA is digested within minute in the cytosol by nucleases DNA is delivered to the nucleus by unknown mechanisms Not clear whether DNA is delivered into the nucleus as a particulate (protected) or naked Methods: - calcium phosphate - positively charged polymers - liposome

Question 26

What are the main 3 chemical methods of transfections?

Answer 26

Methods: - calcium phosphate - positively charged polymers - liposome

Question 27

Explain the calcium phosphate transfection

Answer 27

Use calcium phosphate to precipitate the DNA Precipitates DNA added to cell media and eventually the cells will uptake it by endocytosis

Question 28

What are the advantages and disadvantages of calcium phosphate transfection?

Answer 28

Advantages - very simple - inexpensive - we’ll established - works for most cell types Disadvantages - works for adherent cells only - skill and experience required to get good precipitates - not particularly efficient in some cell types - many cells types do not like adherent precipitates - difficult to automate or perform as a high throughput method

Question 29

How is transfection done using positively charged polymers?

Answer 29

Polymer DIETHTLAMINOETHYL modified dextran used DNA adheres to the DEAE dextran and remains soluble The complex adheres to cells and is taken up by endocytosis

Question 30

What are the advantages and disadvantages of transfection using positively charged polymers?

Answer 30

Advantages - gentle, low toxicity - works well for cells in suspension - high volume transfection Disadvantages - doesn’t work well I many cell types - doesn’t work well for stable transfection - unclear mechanism of action makes optimisation difficult - relatively expensive - low throughput

Question 31

What are liposomes?

Answer 31

Liposomes are vesicle composed of a lipid bilayer surrounding a hollow core into which drugs or other molecules can be loaded for delivery

Question 32

How are unilamellar liposomes formed?

Answer 32

Can be formed from cationic lipids by : Sonication - uneven liposome size, not reproducible Microfluidisation - regular, small, stable liposomes, reproducible

Question 33

How does liposomes mediated transfection work?

Answer 33

DNA is mixed with liposomes and binds electrostatically Liposome/DNA complexes enter cells by fusion with plasma Encapsulate gene in liposome which then fuses with plasma membrane and uptakes through endocytosis by cell

Question 34

What are the advantages and disadvantages of liposome mediated transfection?

Answer 34

Advantages - very simple to perform and optimise - anyone can do it - easy to automate, high throughput - reliable and reproducible - stable and transient assays work well - works well with many cell types and also in vivo, either adherent or non adherent cells Disadvantages - many formulations require use of serum free or serum reduce medium for good efficiency ( all types of neutral lipids) - some formulations are unstable to oxygen - variable toxicity necessitates carful optimisation for many types ( eg lypofectamine) - very expensive to but ( but almost free to make)

Question 35

What is electroporation?

Answer 35

Uses electrical field that transiently permeabilises the membrane during this period, the foreigner material can enter the cells

Question 36

How is electroporation carried out?

Answer 36

Cells and DNA mixed and places into a cuvette between two electrode plates High dc voltage applied as a pulse Optimised by altering voltage, pulse length, wave form. During electroporation electro pulse applied creating gaps in cell membrane, temporary, through pore DNA can pass through Dna suspended in conductive solution Close circuit Short pulse lasting milliseconds applied distrusting bilayer As electric potential across cell membrane rises due to the voltage applied allows charged particles to pass through

Question 37

What are the advantages and disadvantages of electroporation ( non chemical transfection methods)?

Answer 37

Advantages - high transfection efficiency - effective for stable transfection Disadvantages - only works for cells in suspension - devices for transferring adherent cells are tricky and cumbersome to clean - required optimisation as cell permeability can lead to cell damages - experienced users are required - very sensitive to salt concentrations - expensive

Question 38

How is viral transfection or transduction carried out?

Answer 38

Happen naturally when virus transfers genetic material from one bacteria to another Transferring foreign DNA into a cell using a virus

Question 39

When and why is viral transfection used and talk about it’s efficiency

Answer 39

- used for hart to transfer cell types - commonly used in clinical research - often 100% transfection efficiency - viruses evolved to efficiently introduce nuclei acid into cells - can be used for in vivo transfection - engineered for maximum infection but replication defective by mutation / deletion of gag, pol, Env

Question 40

Give examples of viral transfection cells and why they’re used

Answer 40

Adenoviruses, retroviruses - used extensively for gene delivery in mammalian cell cultures Baculovirus - expression in insect cells for Pepsi ting recombinant glycoproteins or membrane proteins

Question 41

What is a vaccine?

Answer 41

biological preparation that provide active acquired immunity to a particular disease Often made from weakened, inactivated or killed forms of microbes, toxins or one of its surface proteins

Question 42

How is a virus produced?

Answer 42

Not very different from antibody production The virus replicates inside cells and the cell environment is the ‘culture enviroment’ (Culture within a culture) Viruses can be produced in eggs ( influenza) or in mammalian cell cultures

Question 43

How are vaccines created in embryo ages eggs?

Answer 43

1) Fertilised eggs held in incubators (9-11 days) 2) inculcation with specific influenza virus 3) incubation for 48196 hours at 33-36*c 4) pre chilling in refrigerator 5) harvest of allantonic fluid 6) purification and virus inactivation (Approx 15ug viral vaccine produces from 1 or 2 eggs ( single human dose))

Question 44

Why are embryonated eggs used for vaccine production?

Answer 44

Eggs are perfect natural mini bioreactors ( nutrient rich, aseptic) Many viruses grow very well in ebryonated chicken eggs Robust yield enables use for reaserch and vaccine production

Question 45

Give 5 examples of vaccines and what cells they use

Answer 45

Flucelvax (us) / optaflu(eu) (Novartis) - first mammalian cell based vaccine against the influenza virus (2012) produced in MDCK cells Polio - in vero cells or human diploid fibroblasts Rabies - in vero cells Measles, mumps - in chicken embryo cells Rubella, chicken pox - in human diploid lung fibroblasts

Question 46

How are vaccines produced when creating it using a mammalian cell culture?

Answer 46

choose cell line of interest example - vero cells Two phases Growth phase - objective is to increase mammalian cell biomass once preferred cell density reached they are infected with viral particles Production phase - objective is to maximise the conversion of the mammalian cell biomass to viable viral biomass Disposable bioreactors usually used 2 types can be used either horizontal on orbital shakers or vertical bags have impeller in them to ensure good mixing

Question 47

What cell types is most suitable for viral growth for large scale productions?

Answer 47

Anchorage dependant cell growth with micro-carrier cultures often used

Question 48

Why use animal cell cultures for viral vaccine production?

Answer 48

Animal viruses propagated by cultured cells at relatively high density

Question 49

How is virus production maximised?

Answer 49

Viral inoculation is accompanied by a feeder to keep cells alive and maximise virus production

Question 50

What is polio?

Answer 50

Poliomyelitis or infantile paralysis Caused by the polio virus Transmission by oral faecal route Multiplies in throat and intestine Invaded blood and lymph Sub clinical - mild symptoms in 95% of people Clinical - non paralytic polio , paralytic polio 1% of cases

Question 51

Explain the properties of the polio virus and it’s associated timeline

Answer 51

2 types of vaccine - an inactivated poliovirus given by injection - a weakened poliovirus given by mouth Developed and tested in 1952 released 1955 Cultured live polio virus in monkey kidney epithelial cells (vero) Virus harvested and killed by formaldehyde Injected vaccine - provides igG mediated immunity to prevent viremia - 60-90% protection I’m 1957 Albert Savin Sabin developed a live attenuate polio vaccine by passaging poliovirus through monkeys - oral vaccine that given intestinal mucosal immunity - occasionally mutates back and gives people polio

Question 52

What’s an antibiotic?

Answer 52

Chemical non toxic compounds used to treat infections caused primarily by bacteria.

Question 53

What’s a natural antibiotic?

Answer 53

True antibiotic - produced naturally by fungi or bacteria to selectively inhibit the growth of others ( chrysogenum produces penicillin

Question 54

What’s a semi synthetic antibiotic?

Answer 54

Chemically modified natural antibiotics ( ampicillin)

Question 55

What’s a totally synthetic antibiotic?

Answer 55

Manufactured antibiotic like trimethoprim

Question 56

Describe the timeline for the discovery of penicillin

Answer 56

1928 : Alexander Fleming noticed a secreted anti microbial produced by the penicillium fungus. This compound was penicillin Between 1928 and 1945 Howard Florey and Ernst chain devised methods to produce large quantities of penicillin - the first B-road spectrum antibiotic with low toxicity and very good activity 1940s : penicillin was first introduced at large scale (20l) and purified by Howard Florey and Ernst chain Fleming chain and Florey shared the Nobel prize for medicine in 1945

Question 57

How do antibiotics work?

Answer 57

They work in one of 2 ways : Bactericidal: kills the bacteria by preventing it to make a cell wall Bacteriostatic : prevents bacteria from dividing by interfering with processing like DNA replication, metabolism ( enzyme activity) and protein production

Question 58

What are broad and narrow spectrum antibiotics?

Answer 58

Broad spectrum - kill a wide range of bacteria ( penicillin) Narrow spectrum - kill a specific types or group of bacteria ( isoniazid)

Question 59

What are the 4 mechanism of action of antibiotics?

Answer 59

Cell wall synthesis inhibitors - b lactams - glycopeptides - cycloserine, bacitracin Targeting cell membrane - polymyxin Inhibit nucleic acid synthesis (DNA replication) - sulphinamides - inhibits cell division not kill Inhibit protein synthesis - stops replication - tetracyclines

Question 60

What was antibiotic production like in 1945?

Answer 60

Mass production was possible Yield was 1g/l of penicillin Not great yield Biggest discovery I’m medicine

Question 61

What does antibiotic production look like currently?

Answer 61

Same fungus used Yields 50g/L of penicillin Culture medium - lactose, other sugars, source of nitrogen - yeast extract Fermented in fed batch mode ( to prolong stationary phase of the cells and increase production)

Question 62

What phase of the growth cycle does penicillin production occure?

Answer 62

Penicillin is a secondary metabolite so is produced during the stationary phase of cell growth

Question 63

Why is penicillin modified?

Answer 63

Penicillin G can be enzymatically modified to make a variety of other penicillins with slightly different properties ( penicillin O, ampicillin, amoxicillin) Depending on what properties are required the penicillin is adjusted accordingly

Question 64

Explain the downstream bioprocessing of antibiotics m

Answer 64

Required to increase purity 1) involves filtration to remove solids 2) the filtered broths is acidified and extracted with an organic solvent Extract is then stripped with buffer to isolate a concentrate 3) pH of concentrate is adjusted with acid and product is purified by second extraction into organic solvent 4) final polishing consists of vaccine concentration, crystallisation, and drying of crystalline penicillin

Question 65

What is cephalosporin?

Answer 65

A b lectim antibiotic used to treat wide range of infections from grim positive to grim negative bacteria Broad spectrum antibiotic

Question 66

What are the culture conditions for celphalosporin?

Answer 66

Media - source carbon ( glucose, starch, sucrose), source of nitrogen (meat extract, peptone) Growth conditions - pH 6-7 temp 24-28, for 4-10 days High oxygen consumption - compressed filter sterilised air is supplied Fermentation - fed batch mode to prolong stationary phase Downstream RIPP

Question 67

How does antibiotic resistance occur?

Answer 67

High number of bacteria A few of them are resistant to antibiotics Antibiotics kill bacteria causing illness as well as good bacteria protecting body from infection The resistant bacteria now have preferred conditions to grow and take over Bacteria can transfer their drug resistance to other bacteria causing more problems

Question 68

How long can it take to bring a new antibiotic onto the market?

Answer 68

Up to 15 years

Question 69

Explain the steps of the hybridoma technique for producing monoclonal antibodies

Answer 69

1) immunisation of mouse 2) B cell isolation from spleen 3) in parallel, the cultivation of myeloma cells ( lack the HGRT gene) 4) myeloma and B cells fusion ( electro fusion or chemically 5) culture fused in HAT medium for 10-14 days. Unfused myeloma cells will die in the HAT medium. Unfused B cells die because they have a short life span 6) dilution of the hybridoma suspension until one cell per well 7) cell line screening for antibodies of appropriate specificity ( by using elsa) 8) in vitro (a) or in vivo (b) multiplication 9) harvesting of MAbs